【摘要】 目的探讨"10-23"脱氧核酶("10-23"DZ)抑制突变型p53基因表达的效应。方法设计合成针对mp53(R273H,CGT﹥CAT)的"10-23"DZ:p53DZ7、p53DZ9、p53DZ11及硫代修饰物p53DZ11-s。在无细胞体系观察"10-23"DZ对p53RNA的切割效应。经脂质体将DZ转染进HT29结肠癌细胞株,RT-PCR检测各种DZ对HT29细胞mp53mRNA的影响;免疫细胞化学及ImageProPlus4.5图像分析系统检测mp53蛋白,观察各种DZ及ASO对mp53蛋白表达的影响。结果p53DZ11和p53DZ11-s在无细胞体系能有效切割mp53RNA,而对wp53RNA的作用弱。在细胞内p53DZ11-s在浓度为250nM时,能下调HT29细胞mp53mRNA水平和mp53蛋白表达。结论针对mp53的273突变点所设计的p53DZ11、p53DZ11-s在细胞外均能有效切割mp53RNA,在HT29细胞内p53DZ11-s也能抑制mp53mR-NA及mp53蛋白的表达。更多还原

【Abstract】 OBJECTIVE To investigate the inhibitory effect of mutant p53 gene expression by "10-23" DNAzyme in vitro. METHODS Three "10-23" DNAzymes: p53DZ7, p53DZ9 and p53DZ11 targeting mp53 RNA (R273H, CGT﹥CAT) were designed. In order to increase the stability of the DZ, the first two bases at the two ends were substituted with phosphorothioates: p53DZ11-s. The RNA cleavage activities of DZ were measured in cell free system. DZs were transfected into HT29 cells by OligofectamineTM Reagent. RT-PCR was performed for evaluating the inhibitory activity of DZs on mp53 mRNA expression, and immunocytochemical methods and Image Pro Plus 4.5 were used to detect the p53 protein expression on HT29 cell. RE-SULTS p53DZ11 and p53DZ11-s were able to cut mp53 RNA at the expected site effectively, while the rates p53DZ11 and p53DZ11-s cleaving the mp53 were low. In HT29 cell, p53DZ11-s was capable of decreasing the mp53 mRNA expression obviously, at 250nM of DZ p53DZ11-s and p53DZs-cho can inhibit the p53 protein expression of HT cell obviously. CONCLU-SION In cell free system, p53DZ11 and p53DZ11-s targeting mp53 (R273H) RNA could cleave mp53 RNA. The expression of mp53 mRNA and mp53 protein was inhibited by p53DZ11-s in HT29 cell.更多还原